RESUMO
Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel three-dimensional (3D) tracking of multiple chromosomal loci of choice over many thousands of frames on various timescales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking both of fast dynamics (50 Hz) and over timescales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent α and a transition to more-isotropic dynamics in 3D after such treatment. We think that in the future our methodology will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.
Assuntos
Cromatina , Cromossomos , Animais , Anisotropia , Difusão , Regulação da Expressão Gênica , MamíferosRESUMO
Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion.
Assuntos
Actinina/química , Células Epiteliais/metabolismo , Miosinas/química , Neurônios/metabolismo , Engenharia de Proteínas/métodos , Espectrina/química , Actinina/genética , Actinina/metabolismo , Animais , Avena , Linhagem Celular , Chara , Galinhas , Clonagem Molecular , Dictyostelium , Células Epiteliais/citologia , Células Epiteliais/efeitos da radiação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Luz , Modelos Moleculares , Movimento (Física) , Miosinas/genética , Miosinas/metabolismo , Neurônios/citologia , Neurônios/efeitos da radiação , Óptica e Fotônica/métodos , Cultura Primária de Células , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espectrina/genética , Espectrina/metabolismo , NicotianaRESUMO
Segmenting cell nuclei within microscopy images is a ubiquitous task in biological research and clinical applications. Unfortunately, segmenting low-contrast overlapping objects that may be tightly packed is a major bottleneck in standard deep learning-based models. We report a Nuclear Segmentation Tool (NuSeT) based on deep learning that accurately segments nuclei across multiple types of fluorescence imaging data. Using a hybrid network consisting of U-Net and Region Proposal Networks (RPN), followed by a watershed step, we have achieved superior performance in detecting and delineating nuclear boundaries in 2D and 3D images of varying complexities. By using foreground normalization and additional training on synthetic images containing non-cellular artifacts, NuSeT improves nuclear detection and reduces false positives. NuSeT addresses common challenges in nuclear segmentation such as variability in nuclear signal and shape, limited training sample size, and sample preparation artifacts. Compared to other segmentation models, NuSeT consistently fares better in generating accurate segmentation masks and assigning boundaries for touching nuclei.
Assuntos
Núcleo Celular/fisiologia , Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Algoritmos , Artefatos , Biologia Computacional , Células HeLa , Humanos , SoftwareRESUMO
Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sistemas CRISPR-Cas , Cálcio/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Oncogenes/genética , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
Women with dense breasts have an increased lifetime risk of malignancy that has been attributed to a higher epithelial density. Quantitative proteomics, collagen analysis, and mechanical measurements in normal tissue revealed that stroma in the high-density breast contains more oriented, fibrillar collagen that is stiffer and correlates with higher epithelial cell density. microRNA (miR) profiling of breast tissue identified miR-203 as a matrix stiffness-repressed transcript that is downregulated by collagen density and reduced in the breast epithelium of women with high mammographic density. Culture studies demonstrated that ZNF217 mediates a matrix stiffness- and collagen density-induced increase in Akt activity and mammary epithelial cell proliferation. Manipulation of the epithelium in a mouse model of mammographic density supported a causal relationship between stromal stiffness, reduced miR-203, higher levels of the murine homolog Zfp217, and increased Akt activity and mammary epithelial proliferation. ZNF217 was also increased in the normal breast epithelium of women with high mammographic density, correlated positively with epithelial proliferation and density, and inversely with miR-203. The findings identify ZNF217 as a potential target toward which preexisting therapies, such as the Akt inhibitor triciribine, could be used as a chemopreventive agent to reduce cancer risk in women with high mammographic density.
Assuntos
Neoplasias da Mama , Glândulas Mamárias Humanas , Proteínas Oncogênicas/metabolismo , Transativadores/metabolismo , Adulto , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Método Duplo-Cego , Feminino , Humanos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Neoplásico/metabolismo , Fatores de RiscoRESUMO
By examination of the cancer genomics database, we identified a new set of mutations in core histones that frequently recur in cancer patient samples and are predicted to disrupt nucleosome stability. In support of this idea, we characterized a glutamate to lysine mutation of histone H2B at amino acid 76 (H2B-E76K), found particularly in bladder and head and neck cancers, that disrupts the interaction between H2B and H4. Although H2B-E76K forms dimers with H2A, it does not form stable histone octamers with H3 and H4 in vitro, and when reconstituted with DNA forms unstable nucleosomes with increased sensitivity to nuclease. Expression of the equivalent H2B mutant in yeast restricted growth at high temperature and led to defective nucleosome-mediated gene repression. Significantly, H2B-E76K expression in the normal mammary epithelial cell line MCF10A increased cellular proliferation, cooperated with mutant PIK3CA to promote colony formation, and caused a significant drift in gene expression and fundamental changes in chromatin accessibility, particularly at gene regulatory elements. Taken together, these data demonstrate that mutations in the globular domains of core histones may give rise to an oncogenic program due to nucleosome dysfunction and deregulation of gene expression. SIGNIFICANCE: Mutations in the core histones frequently occur in cancer and represent a new mechanism of epigenetic dysfunction that involves destabilization of the nucleosome, deregulation of chromatin accessibility, and alteration of gene expression to drive cellular transformation.See related commentary by Sarthy and Henikoff, p. 1346.This article is highlighted in the In This Issue feature, p. 1325.
Assuntos
Histonas/genética , Mutação , Neoplasias/genética , Oncogenes , Alelos , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Histonas/química , Histonas/metabolismo , Humanos , Mutação de Sentido Incorreto , Neoplasias/metabolismo , Nucleossomos/metabolismo , Multimerização Proteica , Leveduras/genética , Leveduras/metabolismoRESUMO
The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.
Assuntos
Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Nucleossomos/metabolismo , Sequência de Bases , Linhagem Celular , Cromatina , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Ligação Proteica , Domínios ProteicosRESUMO
The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.
Assuntos
Transformação Celular Neoplásica/patologia , Células Epiteliais/patologia , Glândulas Mamárias Humanas/patologia , Células Acinares/patologia , Animais , Cápsulas , Carcinogênese/patologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Hidrogéis/química , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos SCID , Análise de Sequência de RNA , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
While cells within tissues generate and sense 3D states of strain, the current understanding of the mechanics of fibrous extracellular matrices (ECMs) stems mainly from uniaxial, biaxial, and shear tests. Here, we demonstrate that the multiaxial deformations of fiber networks in 3D cannot be inferred solely based on these tests. The interdependence of the three principal strains gives rise to anomalous ratios of biaxial to uniaxial stiffness between 8 and 9 and apparent Poisson's ratios larger than 1. These observations are explained using a microstructural network model and a coarse-grained constitutive framework that predicts the network Poisson effect and stress-strain responses in uniaxial, biaxial, and triaxial modes of deformation as a function of the microstructural properties of the network, including fiber mechanics and pore size of the network. Using this theoretical approach, we found that accounting for the Poisson effect leads to a 100-fold increase in the perceived elastic stiffness of thin collagen samples in extension tests, reconciling the seemingly disparate measurements of the stiffness of collagen networks using different methods. We applied our framework to study the formation of fiber tracts induced by cellular forces. In vitro experiments with low-density networks showed that the anomalous Poisson effect facilitates higher densification of fibrous tracts, associated with the invasion of cancerous acinar cells. The approach developed here can be used to model the evolving mechanics of ECM during cancer invasion and fibrosis.
Assuntos
Carcinoma de Células Acinares , Colágeno , Matriz Extracelular , Modelos Moleculares , Proteínas de Neoplasias , Animais , Carcinoma de Células Acinares/química , Carcinoma de Células Acinares/metabolismo , Carcinoma de Células Acinares/patologia , Linhagem Celular Tumoral , Colágeno/química , Colágeno/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , RatosRESUMO
We describe three optical tags, ArrayG, ArrayD and ArrayG/N, for intracellular tracking of single molecules over milliseconds to hours. ArrayG is a fluorogenic tag composed of a green fluorescent protein-nanobody array and monomeric wild-type green fluorescent protein binders that are initially dim but brighten ~26-fold on binding with the array. By balancing the rates of binder production, photobleaching and stochastic binder exchange, we achieve temporally unlimited tracking of single molecules. High-speed tracking of ArrayG-tagged kinesins and integrins for thousands of frames reveals novel dynamical features. Tracking of single histones at 0.5 Hz for >1 hour with the import competent ArrayG/N tag shows that chromosomal loci behave as Rouse polymers with visco-elastic memory and exhibit a non-Gaussian displacement distribution. ArrayD, based on a dihydrofolate reductase nanobody array and dihydrofolate reductase-fluorophore binder, enables dual-color imaging. The arrays combine brightness, fluorogenicity, fluorescence replenishment and extended fluorophore choice, opening new avenues for tracking single molecules in living cells.
Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Linhagem Celular , Cor , Corantes Fluorescentes/síntese química , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Anticorpos de Domínio ÚnicoRESUMO
Contractile cells can reorganize fibrous extracellular matrices and form dense tracts of fibers between neighboring cells. These tracts guide the development of tubular tissue structures and provide paths for the invasion of cancer cells. Here, we studied the mechanisms of the mechanical plasticity of collagen tracts formed by contractile premalignant acinar cells and fibroblasts. Using fluorescence microscopy and second harmonic generation, we quantified the collagen densification, fiber alignment, and strains that remain within the tracts after cellular forces are abolished. We explained these observations using a theoretical fiber network model that accounts for the stretch-dependent formation of weak cross-links between nearby fibers. We tested the predictions of our model using shear rheology experiments. Both our model and rheological experiments demonstrated that increasing collagen concentration leads to substantial increases in plasticity. We also considered the effect of permanent elongation of fibers on network plasticity and derived a phase diagram that classifies the dominant mechanisms of plasticity based on the rate and magnitude of deformation and the mechanical properties of individual fibers. Plasticity is caused by the formation of new cross-links if moderate strains are applied at small rates or due to permanent fiber elongation if large strains are applied over short periods. Finally, we developed a coarse-grained model for plastic deformation of collagen networks that can be employed to simulate multicellular interactions in processes such as morphogenesis, cancer invasion, and fibrosis.
Assuntos
Colágeno/metabolismo , Fenômenos Mecânicos , Animais , Fenômenos Biomecânicos , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Camundongos , Modelos Biológicos , Células NIH 3T3 , Ratos , Esferoides Celulares/metabolismo , Estresse MecânicoRESUMO
Bacterial chemoreceptors organize into large clusters at the cell poles. Despite a wealth of structural and biochemical information on the system's components, it is not clear how chemoreceptor clusters are reliably targeted to the cell pole. Here, we quantify the curvature-dependent localization of chemoreceptors in live cells by artificially deforming growing cells of Escherichia coli in curved agar microchambers, and find that chemoreceptor cluster localization is highly sensitive to membrane curvature. Through analysis of multiple mutants, we conclude that curvature sensitivity is intrinsic to chemoreceptor trimers-of-dimers, and results from conformational entropy within the trimer-of-dimers geometry. We use the principles of the conformational entropy model to engineer curvature sensitivity into a series of multi-component synthetic protein complexes. When expressed in E. coli, the synthetic complexes form large polar clusters, and a complex with inverted geometry avoids the cell poles. This demonstrates the successful rational design of both polar and anti-polar clustering, and provides a synthetic platform on which to build new systems.
Assuntos
Membrana Celular/metabolismo , Forma Celular , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas de Bactérias , Polaridade Celular , Técnicas de Cultura , Proteínas Luminescentes , Metiltransferases/metabolismoRESUMO
Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.
Assuntos
Cromatina/genética , Corantes Fluorescentes/química , Genoma Humano , Compostos Heterocíclicos de 4 ou mais Anéis/química , Sequenciamento de Nucleotídeos em Larga Escala , Transposases/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Elementos de DNA Transponíveis/genética , Epigênese Genética , Citometria de Fluxo , Humanos , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Neutrófilos/metabolismo , Coloração e Rotulagem , Transposases/genéticaRESUMO
Soluble karyopherins of the importin-ß (impß) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impß or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impß at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impß, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impß is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impß or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impßâ¢Nup153 complexes but not those of TRN1â¢Nup153. We propose that impß and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Recuperação de Fluorescência Após Fotodegradação , Células HeLa , Humanos , Microscopia Confocal , Modelos Biológicos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Permeabilidade , Interferência de RNA , beta Carioferinas/genética , Proteína ran de Ligação ao GTP/genéticaRESUMO
Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100-200nm membrane domains containing tens to hundreds of complexes are indicated. Central to our results is that domains of different functional OXPHOS complexes do not co-localize, but ubiquinone diffusion in the membrane is rapid and long-range, consistent with a mobile carrier shuttling electrons between islands of different complexes. Our results categorically demonstrate that electron transport and proton circuitry in this model bacterium are spatially delocalized over the cell membrane, in stark contrast to mitochondrial bioenergetic supercomplexes. Different organisms use radically different strategies for OXPHOS membrane organization, likely depending on the stability of their environment.
Assuntos
Transporte de Elétrons , Escherichia coli/metabolismo , Fosforilação Oxidativa , Escherichia coli/enzimologia , Ubiquinona/metabolismoRESUMO
Cells and multicellular structures can mechanically align and concentrate fibers in their ECM environment and can sense and respond to mechanical cues by differentiating, branching, or disorganizing. Here we show that mammary acini with compromised structural integrity can interconnect by forming long collagen lines. These collagen lines then coordinate and accelerate transition to an invasive phenotype. Interacting acini begin to disorganize within 12.5 ± 4.7 h in a spatially coordinated manner, whereas acini that do not interact mechanically with other acini disorganize more slowly (in 21.8 ± 4.1 h) and to a lesser extent (P < 0.0001). When the directed mechanical connections between acini were cut with a laser, the acini reverted to a slowly disorganizing phenotype. When acini were fully mechanically isolated from other acini and also from the bulk gel by box-cuts with a side length <900 µm, transition to an invasive phenotype was blocked in 20 of 20 experiments, regardless of waiting time. Thus, pairs or groups of mammary acini can interact mechanically over long distances through the collagen matrix, and these directed mechanical interactions facilitate transition to an invasive phenotype.
Assuntos
Células Acinares/patologia , Neoplasias da Mama/fisiopatologia , Comunicação Celular/fisiologia , Glândulas Mamárias Humanas/citologia , Células Acinares/fisiologia , Células Acinares/ultraestrutura , Linhagem Celular Tumoral , Separação Celular , Colágeno , Escherichia coli , Feminino , Humanos , Estimativa de Kaplan-Meier , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia de FluorescênciaRESUMO
The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF-CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.
Assuntos
Carcinogênese/química , Ativação Enzimática/fisiologia , Microscopia/métodos , Imagem Molecular/métodos , Multimerização Proteica/fisiologia , Transdução de Sinais/fisiologia , Quinases raf/química , Animais , Linhagem Celular , Cricetinae , Proteínas ras/metabolismoRESUMO
Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs) embedded in the nuclear envelope. About 30 different proteins (nucleoporins, nups) arrange around a central eightfold rotational axis to build the modular NPC. Nup188 and Nup192 are related and evolutionary conserved, large nucleoporins that are part of the NPC scaffold. Here we determine the structure of Nup188. The protein folds into an extended stack of helices where an N-terminal 130 kDa segment forms an intricate closed ring, while the C-terminal region is a more regular, superhelical structure. Overall, the structure has distant similarity with flexible S-shaped nuclear transport receptors (NTRs). Intriguingly, like NTRs, both Nup188 and Nup192 specifically bind FG-repeats and are able to translocate through NPCs by facilitated diffusion. This blurs the existing dogma of a clear distinction between stationary nups and soluble NTRs and suggests an evolutionary relationship between the NPC and the soluble nuclear transport machinery. DOI:http://dx.doi.org/10.7554/eLife.00745.001.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Modelos Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Relação Estrutura-AtividadeRESUMO
The mechanisms of enzyme activity on solid substrates are not well understood. Unlike enzyme catalysis in aqueous solutions, enzyme activity on surfaces is complicated by adsorption steps and structural heterogeneities that make enzyme-substrate interactions difficult to characterize. Cellulase enzymes, which catalyze the depolymerization of cellulose, show binding specificities for different cellulose surface morphologies, but the influence of these specificities on the activity of multienzyme mixtures has remained unclear. We developed a metric to quantify binding-target arrangements determined by photoactivated localization microscopy, and we used that metric to show that combinations of cellulases designed to bind within similar but nonidentical morphologies can have synergistic activity. This phenomenon cannot be explained with the binary crystalline or amorphous classifications commonly used to characterize cellulase-binding targets. Our results reveal a strategy for improving the activity of cellulolytic mixtures and demonstrate a versatile method for investigating protein organization on heterogeneous surfaces.